Anticonvulsant Activity of Hydroalcoholic Extract and Aqueous Fraction of Ebenus stellata in Mice.

Objective(s) Fabaceae is the third largest family of flowering plants. Lack of essential oils in the plants of this family can be considered as an advantage and can favor them in search for safe and antiepileptic medicines. The effects of Fabacea family plants including Ebenus stellata (E. stellata), Sophora alopecuroides and Caesalpinia gilliiesii were evaluated in pentylenetetrazole (PTZ) and maximal electroshock (MES) seizure tests. Materials and Methods The hydroalcoholic extracts were obtained by percolation of 100 g aerial parts of each plant in 900 ml ethanol 80%. Acute toxicity of the extracts was assessed. Non-toxic doses of the extracts were injected to the mice intraperitoneally (i.p.) and occurrence of clonic seizures induced by PTZ (60 mg/kg, i.p.) or tonic seizures induced by MES (50 mA, 50 Hz, 1 sec), were monitored up to 30 min after each administration. The anticonvulsant extract was then fractionated by dichloromethane and water. Phytochemical screening of the effective extract was also carried out by thin layer chromatography to verify active constituents. Results Among the extracts used, only E. stellata had no toxicity and inhibited clonic seizures in a significant and dose-dependent (3-7 g/kg) manner with ED50 of 4 g/kg. Fractionation of the extract resulted in dose-dependent (1-5 g/kg) anticonvulsant activity, which was observed in aqueous fraction with ED50 of 1.74 g/kg. Phytochemical screening revealed the presence of terpens/sterols, alkaloids, flavonoids, tannin and saponins in the extract. Conclusion The presence of anticonvulsant compounds in E. stellata suggests further activity-guided fractionation and analytical studies to find the potential of this plant as a source of anticonvulsant agents.


Introduction
Epilepsy is the third most common neurological disorder after stroke and Alzheimer's disease (1). Current available anticonvulsant drugs are able to efficiently control epileptic seizures in about 50% of the patients; 25% of the cases may show improvement, whereas the rest of the patients do not benefit significantly (2). Furthermore, undesirable side effects of the drugs used clinically often render treatment difficult; so that a demand for new types of anticonvulsants exists. One of the approaches to search for new antiepileptic drugs is the investigation of naturally-occurring compounds, which may belong to new structural classes.
Essential oils often have high toxicity and narrow therapeutic indices. Furthermore, their particular chemical structure has low potential for modification, which renders them unsuitable candidates for drug design. Most of the plants of Fabaceae family have no or negligible amount of the essential oils. This can be considered as an advantage and can favor the plants of this family in search for safe and effective medicines pertaining to new structural classes.
In this study the possible anticonvulsant and toxic effects of three plants of Fabaceae family including Ebenus stellata, Sophora alopecuroides and Caesalpinia gilliiesii, were evaluated in mice.

Plant materials
The plants were collected in May 2008. E. stellata was collected from Maharloo lake in southeast of Shiraz. C. gilliiesii was collected from Karaj. S. alopecuroides was collected from sides of Natanz on Isfahan road. The plants were authenticated and the voucher specimens (No.74-8, 74-87 and 85-6, respectively) were deposited in the Herbarium of Pasteur Institute of Iran, Tehran.

Extract preparation
One hundred grams of air-dried aerial parts of each plant were grounded and extracted at the room temperature for 48 hr by percolation method using 80% ethanol (900 ml). The extracts were then concentrated with a rotary evaporator apparatus (IKA-RV 05 basic, Germany) at temperature not exceeding 50 °C. The yields of the extracts were 45% (w/w). The extracts were stored at 4 °C throughout experiments.

Fractionation
The crude extract of E. stellata was suspended in 200 ml distilled water and extracted with dichloromethane (three times, 150 ml each). The dichloromethane and the aqueous fractions were collected separately, dried by rotary evaporator at 40 °C and stored at 4 °C throughout experiments ( Figure 1).

Animals
A total number of 350 male NMRI mice (20-28 g, Pasteur Institute of Iran) were used. The animals were housed in standard cages (ten mice in each cage) with free access to food (standard laboratory rodent's chow) and water. The animal room temperature was maintained at 23±1 °C with a 12 hr light/12 hr dark cycle (light on from 06:00 a.m.). The study was approved by the Ethics Committee of Pasteur Institute of Iran and conforms to the European Communities Council Directive of 24 November 1986 (86/609/EEC). All animal experiments were carried out in such a way to minimize the number of animals and their suffering. Each animal was tested once. All injections were done intraperitoneally (i.p.) in volume of 0.1 ml/10 g of mice body weight.

PTZ-induced seizures
The minimal i.p. dose of PTZ at which 99% of the animals showed general clonus was determined by a dose-percent effect curve (18). General clonus was considered as the criteria of clonic seizure, which was characterized by clonus of four limbs with transient loss of righting reflex (19). The extracts (C. gilliiesii: 0.5 g/kg, S. alopecuroides: 0.2 g/kg; E. stellata: 2, 3, 5, 6 and 7 g/kg) and fractions (f1: 0.5, 1, 3 and 5 g/kg, f2: 3 and 6 g/kg), the solvent of the extracts and the fractions Tween 80 (25%): DMSO (2:1, v/v) 10 ml/kg, as control), ethosuximide (150 mg/kg, as positive control) and saline (10 ml/kg, as control) were injected to the mice (sixteen different groups, ten animals in each group). After 30 min, PTZ (60 mg/kg) was injected to the animals. If no general clonus occurred during a 30-min period of observation, the animals were considered protected.

MES-induced seizure
Electro-convulsive shock, inducing hind limb tonic extension (HLTE) in 99% of the animals (18) was determined by a current intensitypercent effect curve. The electrical stimulus (50 mA; 50 Hz; 1-sec duration) was applied (15) through ear-clip electrodes (using a stimulator apparatus (MGH-777, Development of Electronic Industry, Iran). Six groups of 10 mice, each were pretreated i.p. with the different doses of the extracts (C. gilliiesii: 0.5 g/kg, S. alopecuroides: 0.2 g/kg; E. stellata: 7 g/kg), phenytoin (25 mg/kg, as positive control), saline (10 ml/kg, as control) and the solvent of the extracts (10 ml/kg, as control). After 30 min the animals received transauricular electroshock. If no HLTE was observed within 10 sec after delivery of the electroshock, the animals were considered protected.

Data analysis
The dose of the extract required to produce an anticonvulsant effect (ED 50 ) in 50% of the animals was calculated by the method of Litchfield and Wilcoxon (18) using a commercial computer program (GRAPHPAD INSTAT 3, version 2003). Data obtained from convulsive tests were expressed as the percentage of the animals showing convulsions and Fisher's exact test was used to analyze the data. P-value less than 0.05 was the critical criterion for statistical significance.

Mortality
The crude extracts of S. alopecuroides and C. gilliiesii showed lethal effects at the doses of  (Table 1).

Anticonvulsant activity
The hydroalcoholic extracts of S. alopecuroides and C. gilliiesii up to the maximum non-toxic doses did not show any anticonvulsant effect against seizures induced by PTZ and MES (Tables 2 and 3). The extract of E. stellata up to the dose of 7 g/kg did not show any anticonvulsant effect against tonic seizures induced by MES (Table 3). It however inhibited clonic seizures induced by PTZ and ED 50 of 4 g/kg was obtained for the extract (Figure 2, Figure 3).
Fractionation of E. stellata crude extract by dichloromethane and water led to increase in anticonvulsant potency that was observed in aqueous phase and ED 50 of 1.74 g/kg was obtained (Figure 2, Figure 3).

Preliminary phytochemical analysis
The constituents of the crude extract of E. stellata are demonstrated in Table 4. The extract contains triterpens/sterols, flavonoids, alkaloids, saponins, and tannin. Coumarins, anthrones, valepotriates, anthraquinones and essential oil were absent in the extract.

Discussion
PTZ and MES are the most commonly used preliminary tests for screening of potential anticonvulsant drugs. The MES test is considered to be a predictor of likely therapeutic efficacy against generalized tonic-clonic seizures whereas the PTZ test represents a valid model for human generalized myoclonic and also absence seizures (19).
Fabaceae, which is the third largest family of flowering plants, has been widely studied in search for safe and effective antiepileptic medicines (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). The genus Ebenus belongs to this family and has more than 100 species (3). However, there is no report regarding the biological effects of the plants in this genus, such as E. stellata. The present study is the first report in this regard demonstrating that the crude hydroalcoholic extract of E. stellata possesses protective effect against clonic seizures induced by PTZ. Fractionation of the extract by dichloromethane resulted in 2.5 times augmentation in the anti-seizure potency, as ED 50 of 1.74 g/kg was obtained for the aqueous fraction. This finding indicates that fractionation has been capable of separating the anticonvulsant components from the crude extract. It seems that acceptable potency as well as no toxicity of the aqueous fraction makes it worthy for further studies.
Our results indicated that the active anticonvulsant principle(s) present in E. stellata are polar compounds, since the activity was observed in aqueous fraction and not in the dichloromethane fraction. The phytochemical tests performed in this study showed the presence of triterpens/sterols, alkaloids, flavonoids, tannin, and saponins in the crude extract of E. stellata. The anticonvulsant activity of triterpens (20), flavonoids (21), saponins (22)(23) and alkaloids (24) has been demonstrated previously. Therefore, the anticonvulsant activity of the extract and its aqueous fraction could be attributed to the activity of triterpens, flavonoids and alkaloids present in the plant.
It has been proved that reduction of T-type Ca 2+ currents by drugs such as ethosuximide can prevent seizures induced by PTZ (25).   (26). Furthermore, activation of N-methyl-Daspartate receptor appears to be involved in the initiation and generalization of the PTZinduced seizures (27). Accordingly, drugs that block glutamatergic excitation mediated by NMDA receptor such as felbamate have anticonvulsant activity against PTZ-induced seizures (26). Flavonoids, as one of the major components present in E. stellata, are reported to potentiate GABA-induced currents in native GABA A receptors expressed in cortical neurons (28) and also to selectively modulate GABA A receptor subtypes (29)(30). Moreover, flavonoids block NMDA receptors in a concentration-dependent manner (31)(32).
Alkaloids, the other main component found in E. stellata, have shown anticonvulsant activity against seizures induced by kainic acid, PTZ and bicuculine (33)(34). Terpenoids have also NMDA receptor-blocking (35) and GABA A receptor positive-modulation properties (30). Finally, saponins have been reported to protect NMDA-induced neuronal death via a competitive interaction with the glycine-binding site of NMDA receptors in cultured hippocampal neurons (36). Saponins also block GABA specific transporters selectively, which results in inhibition of GABA uptake (37) and propounds saponin compounds as anticonvulsant agents (38). Collectively, these reports provide some experimental evidence for the involvement of the glutamatergic and GABAergic system in the anticonvulsant action of E. stellata.

Conclusion
The hydroalcoholic extract of E. stellata and its aqueous fraction possess protective effect against PTZ-induced clonic seizures. Acceptable potency and lack of acute toxicity suggest further activity-guided fractionation and analytical studies to explore the anticonvulsant agents present in this plant.